Use of a global assay of coagulation and fibrinolysis to aide in thromboembolism

ABSTRACT

The present invention provides methods for identification of subjects with a rise in plasma coagulability post-diagnosis of thrombotic event (and consequent increased risk for recurrent thromboembolism) as well as for identification of subjects without thrombotic event who exhibit a rise in plasma coagulability for which prophylactic anticoagulation may be warranted for TE prevention, and other methods for identification of perioperative subjects who are at heightened risk for perioperative bleeding and/or for requiring blood transfusion, using the CloFAL assay, to aid in clinical decision making regarding anticoagulant and/or adjunctive clinical management designed to mitigate this heightened thromboembolic risk.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/595,910, filed on Dec. 7, 2017, which is herebyincorporated by reference for all purposes as if fully set forth herein.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under grant no.HL130048-01A1 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Predicting and preventing catastrophic bleeding or excessive clotting(“thrombotic”) episodes in patients with coagulation disorders remains acritical, and largely unrealized, medical challenge. Unlike individualmolecular tests, assays that evaluate net clotting potential or thegeneration of (a) key enzymatic player(s) in the clotting system offerthe potential to assist in the prediction of individual bleeding andthrombotic risk at a given point in time, and even the possibility totailor a specific preventive medical approach to a particular patientbased upon the net balance of his/her clotting system. Historically,such “global assays” have rarely been practical for clinicalapplication. Over the past few years, technological advances have madethe prospect of a clinically useful global assay more tenable. Yet, todate very few such global assays have been designed to evaluate both theclot formation (“coagulation”) and clot breakdown (“fibrinolysis”)abilities of the blood, each of which is an important component of thecoagulation system. Defects in each of these functions have been found,for example, in severe hemophiliacs, as well as in a variety of bleedingand thrombotic disorders.

Despite many scientific advances in recent years to better understandbleeding and thrombotic disorders on the level of gene mutations, suchdiseases continue to cause long-term disability in a significant subsetof patients. The ability to predict catastrophic bleeding or clottingepisodes is an important goal for patients and their treating cliniciansin order to maximize the potential for an enduring high level of patientfunctioning. This goal has remained largely elusive because individualmolecular markers of coagulation do not provide an overall picture of anindividual's hemostatic balance at a given time.

Knowledge of prognostic factors for recurrence in adult and pediatricvenous thromboembolism (VTE) is limited. Elevated plasma levels ofD-dimer (a marker of coagulation activation) following a 6-month courseof anticoagulation in adults with unprovoked VTE are associated with aheightened risk of VTE recurrence, warranting prolonged therapy. Inpatients with provoked VTE, and in young VTE patients more generally,prognostic factors remain largely undefined.

SUMMARY OF THE INVENTION

Recently, using a global assay of clot formation and lysis (CloFAL) onplasma samples collected serially over time, the present inventorobserved a phenomenon of rebound hypercoagulability which we now referto as a paradoxical “marked rise in coagulability in the sub-acuteperiod post-diagnosis of VTE” at 3 months post-diagnosis, relative to4-6 weeks post-diagnosis, in a group of children with provoked VTE whowere enrolled in a single-institution prospective inception cohortstudy.

The data provided by the inventive methods disclosed herein furthermoresuggest that patients who manifest a “marked rise in coagulability” overtime may add prognostic value in the prediction of recurrentthromboembolic events, beyond the prognostic value of an elevatedD-dimer level. In the current standard of care, clinical decision makingon duration of anticoagulation in the treatment of thromboembolismrelies on the clinician's judgment regarding presence or absence ofpersistent hypercoagulability at the time of intended discontinuation ofanticoagulation. Apart from use of the D-dimer test in selectedcircumstances, no laboratory methods exist to assist in thisdecision-making on duration of anticoagulation. In this manner, theinventive methods described herein support new paradigm for assisting inmedical decision-making regarding duration of anticoagulation in thetreatment and secondary prevention of thromboembolism.

Thus, in accordance with an embodiment, the present invention provides amethod for identification or diagnosis of marked rise in coagulabilityin a subject who previously experienced a thrombotic event and underwentcoagulation therapy comprising the steps of: a) analyzing a firstbiological sample taken from a subject at time period of about 4 toabout 6 weeks post cessation of coagulation therapy using the CloFALmethod and generating a CloFAL maximum amplitude value; b) analyzing asecond biological sample taken from a subject at time period of about atleast 2 months post cessation of coagulation therapy using the CloFALmethod and generating a CloFAL maximum amplitude value; c) comparing theCloFAL maximum amplitude value of a) to b); and d) identifying thesubject as being at risk for paradoxical marked rise in coagulabilitywhen the CloFAL maximum amplitude value of b) is at least 50% greaterthan the CloFAL maximum amplitude value of c).

In accordance with a further embodiment, the present invention providesfor a method for prevention or treatment of thrombosis or a thromboticevent a subject who previously experienced a thrombotic event andunderwent anticoagulation therapy and exhibits a marked rise incoagulability wherein a biological sample from the subject was analyzedusing the CloFAL method at time period of about 4 to about 6 weeks, andsubsequently about 3 months, post-diagnosis of VTE, and wherein theCloFAL maximum amplitude value of the sample at about 3 monthspost-diagnosis of VTE was at least 50% greater than the CloFAL maximumamplitude value of the sample at about 4 to about 6 weeks post-diagnosisof VTE.

In accordance with another embodiment, the present invention provides amethod for prevention or treatment of thrombosis or a thrombotic eventin a subject who previously experienced a thrombotic event and underwentanticoagulation therapy who is at risk for a marked rise incoagulability, comprising the steps of: a) analyzing a first biologicalsample taken from a subject at time period of about 4 to about 6 weekspost-diagnosis of VTE using the CloFAL method and generating a CloFALmaximum amplitude value; b) analyzing a second biological sample takenfrom a subject at time period of about 3 months post-diagnosis of VTEusing the CloFAL method and generating a CloFAL maximum amplitude value;c) comparing the CloFAL maximum amplitude value of a) to b); d)identifying the subject as having a marked rise in coagulability whenthe CloFAL maximum amplitude value of b) is at least 50% greater thanthe CloFAL maximum amplitude value of a); and e) administering to thesubject an appropriate antithrombotic therapeutic regimen, includingprolonged duration and/or increased intensity of anticoagulation and/orthe administration of adjunctive immunomodulatory therapies.

In accordance with another embodiment, the present invention provides amethod for identification or diagnosis of who is at risk for a markedrise in plasma coagulability in a subject who previously experienced athrombotic event and underwent coagulation therapy comprising the stepsof: a) analyzing a first biological sample taken from a subject at timeperiod of about 6 weeks+/−2 weeks prior to planned cessation ofanticoagulation therapy using the CloFAL method and generating a CloFALmaximum amplitude value; b) analyzing a second biological sample takenfrom a subject at time of planned cessation (or within about 2 weeks ofpost-cessation) of anticoagulation therapy using the CloFAL method andgenerating a CloFAL maximum amplitude value; c) comparing the CloFALmaximum amplitude value of a) to b); and d) identifying the subject ashaving a marked rise in coagulability and hence being at heightened riskfor recurrent thromboembolism when the CloFAL maximum amplitude value ofb) is at least 50% greater than the CloFAL maximum amplitude value ofa).

In accordance with another embodiment, the present invention provides amethod for identification or diagnosis of who is at risk for a markedrise in plasma coagulability in a subject who previously experienced athrombotic event and underwent anticoagulation therapy comprising thesteps of: a) analyzing a first biological sample taken from a subject attime period of about 6 weeks+/−2 weeks prior to planned cessation ofanticoagulation therapy using the CloFAL method and generating a CloFALmaximum amplitude value; b) analyzing a second biological sample takenfrom a subject at time of planned cessation (or within about 2 weeks ofpost-cessation) of anticoagulation therapy using the CloFAL method andgenerating a CloFAL maximum amplitude value; c) comparing the CloFALmaximum amplitude value of a) to b); and d) identifying the subject ashaving a marked rise in coagulability and hence being at heightened riskfor recurrent thromboembolism when the CloFAL maximum amplitude value ofb) is at least 50% greater than the CloFAL maximum amplitude value ofa); and e) administering to the subject an appropriate antithrombotictherapy regimen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Receiver operating characteristic curves for subacute percentchange (measurements at 3 months vs. 4-6 weeks post-diagnosis of indexVTE) in CloFAL assay parameters.

FIG. 2. Serial CloFAL assay waveforms at 4-6 weeks and 3 monthspost-event in two exemplary children with provoked VTE. In each panel,the pooled normal plasma standard is shown for reference (FACT curve).The patient's samples are run in triplicate and absorbance data is thenaveraged at each time point for the triplicate plasma wells (Cumulativecurve). Panel A: 14 y.o. male with a provoked lower extremity DVT.CloFAL assay maximum amplitude (MA) and AUC₆₀, as measures of globalhypercoagulability, were both elevated acutely (4-6 weeks) butnormalized by the 3 month post-DVT sampling (MA 0.32 and 0.23 at 4-6weeks and 3 months respectively; AUC60 12.9 and 8.8 at 4-6 weeks and 3months respectively). Panel B: 15 y.o. male with a central venouscatheter-associated upper extremity DVT. CloFAL assay shows an increasein MA>50% at the 3 months relative to the 4-6 weeks post-DVT periodconsistent with a marked rise in coagulability (MA 0.25 and 0.45 at 4-6weeks and 3 months respectively).

FIG. 3. A table comprising a number of differing non-limiting examplesof treatment regimens used in connection with the identification of amarked rise in coagulability in a subject, using the methods describedherein.

DETAILED DESCRIPTION OF THE INVENTION

In various embodiments, parameters of clotting and/or fibrinolysisderived from the disclosed methods may be “used for the detection,diagnosis and/or prognosis of various disease states that affecthemostatic balance, such as hemophilia, von Willebrand's disease andother bleeding or prothrombotic conditions. The disclosed methods are ofuse to assess an individual's prothrombotic and/or hemorrhagictendencies in a wide variety of conditions, such as trauma, acutecoronary events/syndromes, cardiac bypass, organ transplantation,intensive care, diagnostic surgical biopsies, or other surgical ormedical procedures.

In accordance with some embodiments, the present invention relates tomethods and compositions for evaluating clot formation and fibrinolysisin a sample from a subject who previously experienced a thrombotic eventand underwent anticoagulation therapy.

In accordance with some other embodiments, the present invention relatesto methods and compositions for evaluating clot formation andfibrinolysis in a sample from a subject who previously experienced athrombotic event but who never received anticoagulation therapy (refusedor had contraindications at the time), and exhibits a rise incoagulability over time by CloFAL assay, and therefore should bereassessed for (or reconsider) initiating anticoagulation.

As used herein, the term “marked rise in coagulability” means that,among subjects who were placed on anticoagulation therapy due to athrombotic event or surgery, a proportion of such patients show a markedincrease over time in CloFAL assay parameters that measure coagulativepotential in plasma. In some embodiments, the CloFAL assay parameterwhich significantly correlate with paradoxical marked rise incoagulability is the maximum amplitude of the CloFAL waveform, or (MA).

As used herein, the term “treatment” or “treating” is an art-recognizedterm which includes curing as well as ameliorating at least one symptomof any condition or disease. Treating includes reducing the likelihoodof a disease, disorder or condition from occurring in an animal whichmay be predisposed to the disease, disorder and/or condition but has notyet been diagnosed as having it; inhibiting the disease, disorder orcondition, e.g., impeding its progress; and relieving the disease,disorder or condition, e.g., causing any level of regression of thedisease; inhibiting the disease, disorder or condition, e.g., impedingits progress; and relieving the disease, disorder or condition, even ifthe underlying pathophysiology is not affected or other symptoms remainat the same level. In accordance with some embodiments, the disease orcondition being treated is related to disease states that affecthemostatic balance, such as hemophilia, von Willebrand's disease andother bleeding or prothrombotic conditions.

“Prophylactic” or “therapeutic” treatment is art-recognized and includesadministration to the host of one or more of the subject compositions.If it is administered prior to clinical manifestation of the unwantedcondition (e.g., disease or other unwanted state of the host animal)then the treatment is prophylactic, i.e., it protects the host againstdeveloping the unwanted condition, whereas if it is administered aftermanifestation of the unwanted condition, the treatment is therapeutic(i.e., it is intended to diminish, ameliorate, or stabilize the existingunwanted condition or side effects thereof).

As used herein, the term “subject” refers to any mammal, including, butnot limited to, mammals of the order Rodentia, such as mice andhamsters, and mammals of the order Logomorpha, such as rabbits. It ispreferred that the mammals are from the order Carnivora, includingFelines (cats) and Canines (dogs). It is more preferred that the mammalsare from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). It ismost preferred that the mammals are of the order Primates, Ceboids, orSimoids (monkeys) or of the order Anthropoids (humans and apes). Anespecially preferred mammal is the human.

In accordance with some embodiments, the subject is a human. In otherembodiments, the subject is a juvenile or adolescent with an age of lessthan 21 years.

In accordance with some embodiments of the present invention, it will beunderstood that the term “biological sample” or “biological fluid”includes, but is not limited to, any quantity of a substance from aliving or formerly living patient or mammal. Such substances include,but are not limited to, blood, serum, plasma, urine, cells, organs,tissues, bone, bone marrow, lymph, lymph nodes, synovial tissue,chondrocytes, synovial macrophages, endothelial cells, and skin. In apreferred embodiment, the fluid is blood or serum.

Thus, in accordance with an embodiment, the present invention provides amethod for identification or diagnosis of a marked rise in plasmacoagulability in a subject who previously experienced a thrombotic eventand underwent anticoagulation therapy, comprising the steps of: a)analyzing a first biological sample taken from a subject at time periodof about 4 to about 6 weeks post-diagnosis of VTE using the CloFALmethod and generating a CloFAL maximum amplitude value; b) analyzing asecond biological sample taken from a subject at time period of about a3 months post-diagnosis of VTE using the CloFAL method and generating aCloFAL maximum amplitude value; c) comparing the CloFAL maximumamplitude value of a) to b); and d) identifying the subject as being atrisk for recurrent VTE when the CloFAL maximum amplitude value of b) isat least 50% greater than the CloFAL maximum amplitude value of a).

In accordance with a further embodiment, the present invention providesfor a method for prevention or treatment of thrombosis or a thromboticevent in a subject who previously experienced a thrombotic event andunderwent anticoagulation therapy who is at risk for a marked rise inplasma coagulability (and has a consequently heightened risk ofrecurrent VTE), wherein a biological sample from the subject wasanalyzed using the CloFAL method at time period of about 4 to about 6weeks, and subsequently approximately 3 months, post-diagnosis of VTE,and wherein the CloFAL maximum amplitude value of the sample atapproximately 3 months post-diagnosis of VTE was at least 50% greaterthan the CloFAL maximum amplitude value of the sample of time period ofabout 4 to about 6 weeks post-diagnosis of VTE.

In accordance with another embodiment, the present invention provides amethod for prevention or treatment of thrombosis or a thrombotic eventin a subject who previously experienced a thrombotic event and underwentanticoagulation therapy who is at risk for a marked rise in plasmacoagulability (and has a consequently heightened risk of recurrent VTE),comprising the steps of: a) analyzing a first biological sample takenfrom a subject at time period of about 4 to about 6 weeks post-diagnosisof VTE using the CloFAL method and generating a CloFAL maximum amplitudevalue; b) analyzing a second biological sample taken from a subject attime period of about 3 months post-diagnosis of VTE, using the CloFALmethod and generating a CloFAL maximum amplitude value; c) comparing theCloFAL maximum amplitude value of a) to b); d) identifying the subjectas being at risk for rebound hypercoagulability when the CloFAL maximumamplitude value of b) is at least 50% greater than the CloFAL maximumamplitude value of a); and e) administering to the subject anappropriate antithrombotic therapy regimen.

In some embodiments, the time for the second or later measurement ofCloFAL maximum amplitude value is at least 3 months post-diagnosis ofVTE. In other embodiments, the time period can be 4, 5, 6, 8, 10, 12months, 18 months, 24 months, or longer after diagnosis of VTE.

In accordance with another embodiment, the present invention provides amethod for identification or diagnosis of who is at risk for a markedrise in plasma coagulability in a subject who previously experienced athrombotic event and underwent coagulation therapy comprising the stepsof: a) analyzing a first biological sample taken from a subject at timeperiod of about 6 weeks+/−2 weeks prior to planned cessation ofanticoagulation therapy using the CloFAL method and generating a CloFALmaximum amplitude value; b) analyzing a second biological sample takenfrom a subject at time of planned cessation (or within about 2 weeks ofpost-cessation) of anticoagulation therapy using the CloFAL method andgenerating a CloFAL maximum amplitude value; c) comparing the CloFALmaximum amplitude value of a) to b); and d) identifying the subject asbeing at risk for a rise in plasma coagulability when the CloFAL maximumamplitude value of b) is at least 50% greater than the CloFAL maximumamplitude value of a).

In accordance with another embodiment, the present invention provides amethod for identification or diagnosis of who is at risk for a markedrise in plasma coagulability in a subject who previously experienced athrombotic event and underwent coagulation therapy comprising the stepsof: a) analyzing a first biological sample taken from a subject at timeperiod of about 6 weeks+/−2 weeks prior to planned cessation ofanticoagulation therapy using the CloFAL method and generating a CloFALmaximum amplitude value; b) analyzing a second biological sample takenfrom a subject at time of planned cessation (or within about 2 weeks ofpost-cessation) of anticoagulation therapy using the CloFAL method andgenerating a CloFAL maximum amplitude value; c) comparing the CloFALmaximum amplitude value of a) to b); and d) identifying the subject asbeing at risk for a rise in plasma coagulability when the CloFAL maximumamplitude value of b) is at least 50% greater than the CloFAL maximumamplitude value of a); and e) administering to the subject anappropriate antithrombotic therapy regimen.

In some embodiments, the time for the second or later measurement ofCloFAL maximum amplitude value is at least 3 months post-cessation ofcoagulation therapy. In other embodiments, the time period can be 1, 2,4, 5, 6, 8, 10, 12 months, 18 months, 24 months, or longer aftercessation of coagulation therapy.

In accordance with the methods disclosed herein, the methods fordetermination of evaluating clot formation, overall coagulability, andfibrinolysis in a sample are encompassed in a method designated as ClotFormation and Lysis (CloFAL) assay, as disclosed in International PatentApplication No. WO2006036744, entitled “METHODS FOR A GLOBAL ASSAY OFCOAGULATION AND FIBRINOLYSIS” and incorporated by reference herein as ifset forth in its entirety.

The CloFAL methods used in the inventive methods are described brieflyherein. A clot is formed in a sample of blood or plasma and thereafterthe clot is lysed. The kinetic parameters for formation and lysis of theclot are determined, preferably using a spectrophotometric assay, toassess the individual's net hemostatic balance at a given time, allowingprothrombotic and hemorrhagic risk assessment. In another embodiment,measured parameters can include the maximum amplitude (MA) ofspectrophotometric absorbance, the time to maximum turbidity (T1), thetime to completion of the first phase of decline in turbidity (T2), andthe area under the curve (AUC) over measured time intervals. From suchmeasurements, the coagulation index (CI) and fibrinolytic index (FI) andmodified Fibrinolytic Index (FI₂) may be determined. CI, FI, AUC and/orindividual measured CloFAL parameters are of use to detect or diagnoseprothrombotic and/or hemorrhagic diseases or conditions and to developtherapeutic treatments tailored to the individual's net hemostaticbalance.

In certain embodiments, involving continuous measurement of clot lysisand clot formation in a sample, the information obtained is morecomprehensive and more directly related to actual physiologicalconditions for clot formation and lysis in the body than presentlyavailable assays. The disclosed methods and compositions allow the rapidand inexpensive assessment of the hemostatic balance in an individualover time.

In one embodiment, clot formation and fibrinolysis may be performed in acontainer or test cell, including but not limited to 96-well microtiterplates, into which a sample (e.g. fresh or freeze-thawed, platelet-poorplasma) and appropriate reagents have been added. An exemplary apparatusof use may include a sample, one or more reagents, buffer, a reagentchamber, and a detection instrument, such as a spectrophotometer. Inmore particular embodiments, the reagents added to the reagent chambermay include small amounts of tissue factor (TF), lipidated TF, and/ortissue-type plasminogen activator (tPA). Where exemplary containersexhibit multiple sample compartments, such as a 96-well plate, thesample may preferably be analyzed in replicates, such as duplicate ortriplicate wells of a 96-well plate. An advantage of the disclosedmethods is that the amount of sample required to assay may be relativelysmall, for example 75 μL of plasma sample per well.

Samples may also include a blank well containing only reagent forcomparison with the test samples. Samples may further comprise one ormore cellular entities, such as white blood cells and/or endothelialcells, in suspension or in a monolayer. The plate may be analyzed in anautomated, thermoregulated (37° C.) spectrophotometer and the course ofclot formation and subsequent lysis may be monitored as continuouschanges in the absorbance of the specimen over a course of time, forexample, over three hours. In a preferred embodiment, optical density at405 nm or dual wavelength OD (405 and 630 nm) may be monitoredcontinuously or at selected frequent time intervals. Thespectrophotometer preferably is interfaced with a computer to permitanalysis of kinetic OD measurements using (a) data analysis program(s).A curve may be generated over the course of the assay reactions thatinclude an initial baseline OD, followed by a progressive rise inoptical density to a point of maximum OD, then completed by aprogressive decline in optical density to baseline. A plasma standard(preferably pooled plasma from healthy individuals) and controls(preferably one normal and one to two abnormal controls each, forcoagulative function and fibrinolytic function) may be runsimultaneously with the clinical/laboratory sample(s) using the sameprotocol.

A clotting curve may be generated whereby coagulation and fibrinolyticparameters of the plasma sample are obtained, relative to asimultaneously run pooled normal subject plasma standard. Specificmeasurements may include the lag time (the time from assay initiation totime to clot initiation, as measured by rise in OD above baseline or aspecified threshold), the maximum amplitude (MA) (maximum OD minusbaseline OD), the time to maximum turbidity (T1), the area under thecurve (AUC) over the course of measured time intervals, and the areaover the curve (AOC) over the course of measured time intervals (e.g.,from T1 to 10-30 minutes thereafter, during the phase of decline in OD.A coagulation index (CI) may be calculated, in one example, as the AUCover the course of the first 30 minutes of an assay, referenced to aplasma standard. A fibrinolytic index (FI) may be calculated, forexample, by relating the ratio of T2 to T1 for a sample as compared to astandard, with a correction factor for differences in maximum OD, asdiscussed below. Alternatively, a modified FI (FI₂) may be calculated bythe area above the curve, or a reciprocal AUC, from T1 to up to T1+30minutes for a sample compared to a standard, with a correction factor asabove. Specimens may be compared between normal controls and patientssuspected of having, or known to have, one or more pathologicconditions, such as hemophilia or other diseases relating to clottingand or clot lysis.

Particular details of exemplary embodiments of CloFAL assays areprovided in the Examples below. However, the skilled artisan willrealize that the concentrations of various reagents and times andtemperatures of reactions may be varied from those specified belowwithout undue experimentation by the person of ordinary skill in theart. Further, where various factors, such as calcium, TF and tPA aredisclosed, such factors may be substituted or supplemented withalternative factors known in the art to exhibit similar activities,within the scope of the claimed methods and compositions.

The CloFAL global assay is reproducible and analytically sensitive todeficiencies and excesses of key components in the coagulation andfibrinolytic systems, as well as to physiologic alterations inhemostasis. The measurement of these parameters may be applied to assesssubjects with known and/or as yet undefined hemorrhagic andprothrombotic conditions.

In one embodiment, any of the combination CloFAL assay results may beanalyzed in an individual suffering from a heart condition. Non-limitingexamples of heart conditions include but are not limited to myocardialischemia, myocardial infarction, acute coronary syndromes,atherosclerotic coronary artery disease, valvular disease, andcongestive heart failure.

In another embodiment, any of the combination CloFAL assay results maybe analyzed in an individual suffering from a prothrombotic condition.Examples of prothrombotic conditions include but are not limited tovenous or arterial thromboembolism, including stroke, as well ashypercoagulable states (in particular, factor V Leiden and prothrombin20210 mutations, antiphospholipid antibodies, anticoagulant deficiency,and elevated levels of procoagulant factors, homocysteine, orlipoproteins).

In certain embodiments, any of the combination CloFAL assay results maybe analyzed in an individual suffering from a bleeding condition, or atrisk for severe perioperative bleeding. Non-limiting examples ofbleeding conditions include the hemophilias and other coagulation factordeficiencies or dysfunctions (including a/hypo/dysfibrinogenemia), vonWillebrand disease, platelet function abnormalities and fibrinolyticabnormalities (e.g., PAI-I deficiency). A non-limiting example ofsettings of risk for severe perioperative bleeding include adolescentswith idiopathic scoliosis undergoing posterior spinal fusion surgery, inwhich overall coagulative capacity in plasma, as measuredpre-operatively using the CloFAL assay coagulation index assists in thepre-operative identification of patients who are at heightened risk forsevere intra-operative bleeding and/or who are at heightened risk forrequiring intra/post-operative blood transfusion.

In yet another embodiment, any of the combination CloFAL assay resultsmay be analyzed in healthy children and adults to assess bleeding and/orprothrombotic risk in the steady state and in times of altered(pathologic or physiologic) coagulation and/or fibrinolysis, includingthe special physiologic states of pregnancy and the neonatal period. Anycombination of CloFAL assay may be used as a pre-operative orpre-treatment screening test on a sample from a test subject; inaddition, any combination of CloFAL assay may be used as apost-operative or post-treatment test on a sample from a test subject.

The CloFAL assay described here was modified from those of He et al.(1999) and Smith et al. (2003). As compared to that by Smith et al.,which evaluates only fibrinolysis, the CloFAL assay permits assessmentof coagulability as well. In the method of Smith et al., clotting isartificially induced immediately in the assay and hence coagulativefunction (i.e., coagulability) is unable to be measured, as isconsistent with that assay's unique purpose and role in assessingfibrinolytic function. Furthermore, when compared to the global assay ofHe et al., the CloFAL assay permits testing with a single reagent toevaluate both coagulation and fibrinolysis, rather than requiring (asdoes that of He et al.) the preparation of two distinct reagents forseparate evaluation of the plasma sample. In addition, unlike the assayof He et al., the CloFAL assay does not require the use of thrombin (akey end-product of the coagulation reactions) among the assay reagents.Neither the method of He et al. nor that of Smith et al. is envisionedor shown to detect a marked rise in plasma coagulability post-diagnosisof VTE, nor to identify those VTE patients who are at risk for recurrentVTE. In the CloFAL assay, frozen plasma aliquots are thawed prior toassay, for example in a 37° C. water bath for three minutes. Comparisonof freeze-thawed versus fresh platelet-poor plasma specimens from thesame individual have revealed no differences in the CloFAL curve. Plasmasamples (fresh or freeze-thawed) can be maintained for up to 30 minutesin an ice-water bath until time of assay. For preparation of reactantsolution, recombinant lipidated human TF (American Diagnostica,Stamford, Conn.; 0.5 μg/mL stock solution prepared according tomanufacturer instruction) and two-chain recombinant tPA (AmericanDiagnostica, Stamford, Conn.; 0.5 mg/mL) are added to a stock solutionof Tris-buffered saline (TBS; 66 mM Tris, 130 mM NaCl, pH=7.0)containing 34 mM CaCl₂, to a concentration of 4 pM and 900 ng/mL,respectively (final concentrations of 2 pM lipidated TF and 450 ng/mLtPA after addition of reactant solution to plasma sample, as describedbelow).

In accordance with another embodiment of the assay, phospholipid and TFcan be used as separate reagents, as an alternative to using lipidatedTF, TBS stock solutions can be stored for up to one month at 4° C., andreconstituted stock solutions of tPA and lipidated TF can be stored forup to one month (and at least 24 hours) at −70° C., for use inpreparation of fresh reactant solution. The reactant solution ismaintained at room temperature until time of assay, not to exceed 30minutes.

Uses of CloFAL Assay for Evaluating and Monitoring Fibrinolytic Capacity

Whether or not cell destruction can be minimized after physiologicalevents such as myocardial infarctions, stroke or gangrene may depend, inpart, upon the existence of pathological or therapeutically inducedfibrinolysis. In order to eliminate or minimize such cell destruction inan individual who has undergone or is undergoing a stroke, heart attackor similar event, it would be useful to rapidly ascertain whether theindividual's clot lysis ability is within a normal range of lyticresponse times. By comparing the individual's specific lytic responsetime to an average lytic response time of a normal, non-pathogenicindividual, or within a given individual over time, a treating physicianmay determine whether the patient's specific lytic response capabilityneeds to be treated or otherwise taken into consideration.

Under conditions in which arterial or venous thrombosis has occurred oris likely to occur, such as during and after surgery, it becomescritical that the treating physician has reliable information availableabout an individual's fibrinolytic processes. For example, pathologicalthrombus formation is especially likely to occur during cardiac surgeryutilizing extra-corporeal passage of blood. Although clotting duringcardiac surgery may be minimized through use of heparin or otheranticoagulants, a surgical patient's natural lytic ability can helpavoid surgical complications by dissolving any pathological thrombi thatform. If a particular surgical patient's lytic ability is impaired, aphysician may elect to administer thrombolytic agents to maintain aparticular level of lytic activity and to avoid the possibility ofpermanent and disabling clot formation occurring during surgery. Tomaintain a desired level of lytic activity, it would be useful to assesswhether the administration of a thrombolytic agent had the desiredeffect upon the surgical patient.

Furthermore, when a deep venous thrombosis or pulmonary embolism isveno-occlusive and/or extensive, compromising venous or pulmonaryfunction or risking chronic venous insufficiency due to venous valvulardamage, thrombolytic therapy may be indicated. Such therapy would bebetter monitored (and its bleeding complications potentially minimized)through use of an assay designed to measure fibrinolytic capacity ofplasma at a given time or within a selected time period, such aspre-treatment, during treatment, or post-treatment.

Generally, in the CloFAL global assay, blood is collected with the childor adult participant at rest in the seated position by atraumaticperipheral venipuncture technique with minimal applied stasis. Samplesare collected into BD Vacutainer, 3.2% buffered sodium citrate,siliconized blood collection tubes (Becton-Dickinson, Franklin Lakes,N.J.) or similar citrated blood collection tubes, with collection of theinitial 1 mL of blood into a discard tube. All specimens are centrifugedfor 15 minutes at 4° C. and 2500×g, and the plasma supernatant is thencentrifuged for an additional 15 minutes to remove any residualplatelets. All samples are aliquoted into 1.5 mL polypropylene long-termfreezer storage tubes with O-ring screw caps (USA Scientific, Ocala,Fla.) or similar long-term freezer storage tubes and stored at −80° C.until time of assay.

The CIoFAL assay permits testing with a single reagent to evaluate bothcoagulation and fibrinolysis. In addition, the CIoFAL assay does notrequire the use of thrombin (a key end-product of the coagulationreactions) among the assay reagents. Frozen plasma aliquots are thawedprior to assay, for example in a 37° C. water bath for three minutes.Plasma samples (fresh or freeze-thawed) can be maintained for up to 30minutes in an ice-water bath until time of assay. For preparation ofreactant solution, recombinant lipidated human TF (American Diagnostica,Stamford, Conn.; 0.5 μg/mL stock solution are prepared according tomanufacturer instruction) and two-chain recombinant tPA (AmericanDiagnostica, Stamford, Conn.; 0.5 mg/mL) are added to a stock solutionof Tris-buffered saline (TBS; 66 mM Tris, 130 mM NaCl, pH=7.0)containing 34 mM CaCl₂, to a concentration of 4 pM and 900 ng/mL,respectively (final concentrations of 2 pM lipidated TF and 450 ng/mLtPA after addition of reactant solution to plasma sample, as describedbelow). TBS stock solutions were stored for up to one month at 4° C.,and reconstituted stock solutions of tPA and TF were stored for up toone month (and at least 24 hours) at −80° C., for use in preparation offresh reactant solution. The reactant solution was maintained at roomtemperature until time of assay, not to exceed 30 minutes.

For each patient sample to be analyzed, 75 μL of freeze-thawed or freshplasma was dispensed into each of three wells in a round-bottom,96-well, Nunc assay plate (Fisher Scientific, Santa Clara, Calif.), andthen pre-warmed at 37° C. for three minutes. Using a multi-tip automatedpipette, 75 μL of reactant solution was added simultaneously to eachwell. The plate was then immediately placed in a thermoregulatedmulti-channel microplate spectrophotometer (for example, PowerWave HT,Bio-Tek Instruments, Winooski, Vt.) for dual kinetic absorbancemeasurements at 405 nm and 630 nm at serial (for example, 45-second)intervals for one or more (for example, 3) hours, following an initialfive-second mixing step prior to the first reading. Thespectrophotometer interfaced with a computer such that all itsoperations, including continuous analysis of delta-absorbance (405 nmminus 630 nm) data using KC4™ PC or other instrument-specific software,may be automated. As shown in FIG. 1, beginning at time zero (T0), acurve is generated over the course of the assay reactions that had aninitial baseline absorbance, followed by a progressive rise inabsorbance to a point of maximum absorbance (achieved at T1), then afirst phase of decline in absorbance (ending at T2, the time point atwhich the slope of decline in absorbance changes by +0.10 mOD/min), andcompleted by a further decline in absorbance to baseline.

The kinetic absorbance data is exported to Microsoft Excel or other dataanalytic software. Using the absorbance data in each triplicate well (orother replicate number of wells) of a given plasma specimen, the maximumamplitude of rise in absorbance is determined (MA=maximum absorbanceminus baseline absorbance, where baseline absorbance was obtained byaveraging the third through eighth kinetic readings). The MA values fromthe replicate wells are then averaged to generate an MA value for thespecimen. T1 and T2 were also obtained, and averaged from the replicatewells to generate T1 and T2 values for the specimen. In one exampleusing the area under the curve (AUC) over the course of the initial 30minutes of the assay, a coagulation index (CI) can be calculated thatrelates this value for the sample to that of the standard run with eachassay (FACT, George King Biomedical, Overland Park, Kans.), as follows:

${CI} = {\frac{\left( {AUC}_{0\mspace{14mu} {through}\mspace{14mu} 30\mspace{14mu} \min} \right)_{sample}}{\left( {AUC}_{0\mspace{14mu} {through}\mspace{14mu} 30\mspace{14mu} \min} \right)_{standard}} \times 100}$

A fibrinolytic index (FI) is calculated by relating the ratio of thetime to completion of the first phase of decline in absorbance (T2) tothe time to maximum absorbance (T1) for the sample as compared to thestandard, with a correction factor for differences in maximum absorbance(MA_(standard)/MA_(sample)), as follows:

${FI} = {\frac{T\; {2/\left( {T\; 1*{MA}} \right)_{sample}}}{T\; {2/\left( {T\; 1*{MA}} \right)_{standard}}} \times 100}$

CI and FI are each reported as averaged values among the replicatewells.A modified FI is calculated as follows (where AOC=area over the curve):

${FI}_{2} = \frac{{AOC}_{{({{T\; 1\mspace{14mu} {through}\mspace{14mu} T\; 1} + {20\mspace{14mu} \min}})}\mspace{11mu} {sample}}}{{MA}_{sample}}$

As an additional index of coagulability, cumulative AUC for the first 60minutes of the assay (AUC₆₀) as a percent of the standard, are alsocalculated. In particular, AUC₆₀ (with calculation provided below) hasbeen observed to be associated with hypercoagulability and thrombotictendency:

${AUC}_{60} = {\frac{\left( {AUC}_{0\mspace{14mu} {through}\mspace{14mu} 60\mspace{14mu} \min} \right)_{sample}}{\left( {AUC}_{0\mspace{14mu} {through}\mspace{14mu} 60\mspace{14mu} \min} \right)_{standard}} \times 100}$

In summary, the CloFAL curve of each plasma specimen is analyzed for MA,T1, T2, CI, FI and/or FI₂, and AUC₆₀.

CloFAL parameters may be measured in age-specific healthy controlsubpopulations for the establishment of normative reference values, andmay also be measured in disease-specific groups at baseline/steady-stateclinical status for the establishment of disease-specific referenceranges in this state. These reference ranges then serve as the basis fordetermination of a given subject's plasma coagulative and fibrinolyticfunctions at a given time via CloFAL assay as normal versus abnormalrelative to healthy and/or similarly-diseased individuals, and ifabnormal, whether indicating a tendency toward hypo- versushypercoagulability and hypo- versus hyperfibrinolysis, and hence atendency toward thromboembolism versus pathological bleeding. Inaddition, a given subject's plasma coagulative and fibrinolyticfunctions at a given time may each be assessed by CloFAL assay asincreased versus decreased relative to another clinically-informativetime point in which that subject's plasma coagulative and fibrinolyticfunctions were, or will be, assess by CloFAL assay, toward thedetermination of relative changes in intra-individual tendency towardthromboembolism versus pathological bleeding.

In accordance with some embodiments, the present inventive methodsprovide a means for identifying or diagnosing a subject with anincreased risk of a thrombotic event and/or marked rise in plasmacoagulability over time. In such instances, the subject is then placedon an appropriate treatment regimen to prevent a new thromboembolism.

As used herein, the term “antithrombotic therapy regimen” can comprise anumber of differing regimens including, but not limited to, an effectivefrequency and intensity of anticoagulation, including oral,subcutaneously injectable, or intravenous anticoagulants. Non-limitingexamples of oral anticoagulants include vitamin K antagonists, directfactor Xa inhibitors, and direct thrombin inhibitors. Non-limitingexamples of subcutaneously injectable anticoagulants include lowmolecular weight heparins and synthetic pentasaccharides. Non-limitingexamples of intravenous anticoagulants include unfractionated heparinand direct thrombin inhibitors. Non-limiting examples of such treatmentregimens used in connection with the assay described herein are shown inthe table in FIG. 3.

In the circumstance of the determination of a marked rise in plasmacoagulability by CloFAL assay during the subacute period post-diagnosisof VTE, examples of potential changes in anticoagulant management aimedat mitigating the heightened risk of recurrent VTE include increasingthe dose of the existing anticoagulant agent, and/or prolonging thecourse of the existing anticoagulant treatment, and/or addingimmunomodulatory therapies to the existing anticoagulant treatment inthe setting of clinical knowledge or clinical suspicion of a systemicinflammatory or autoimmune contribution to the marked rise in plasmacoagulability. Non-limiting examples of such adjunctive immunomodulatorytherapies include corticosteroids, calcineurin inhibitors, inosinemonophosphate dehydrogenase inhibitors, and anti-interleukin monoclonalantibodies,

It will be understood by those of skill in the art, that the inventivemethods also include monitoring the subject post-treatment, and duringfollow-up preventative treatment, until the subject's CloFAL MA levelsare considered to be in the normal range or to have returned to thesubject's baseline (or in the case of the period several monthspost-diagnosis of VTE, to have returned to less than a 50% increase inthe CloFAL MA value obtained at 4-6 weeks post-diagnosis of VTE).

As used herein, the terms “effective” and “effectiveness” includes bothpharmacological effectiveness and physiological safety. Pharmacologicaleffectiveness refers to the ability of the treatment to result in adesired biological effect in the patient. Physiological safety refers tothe level of toxicity, or other adverse physiological effects at thecellular, organ and/or organism level (often referred to asside-effects) resulting from administration of the treatment. On theother hand, the term “ineffective” indicates that a treatment does notprovide sufficient pharmacological effect to be therapeutically useful,even in the absence of deleterious effects, at least in an unstratifiedpopulation. (Such a treatment may be ineffective in a subgroup that canbe identified by the expression profile or profiles.) “Less effective”means that the treatment results in a therapeutically significant lowerlevel of pharmacological effectiveness and/or a therapeutically greaterlevel of adverse physiological effects, e.g., greater liver toxicity.

The following examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter. The synthetic descriptions and specific examples thatfollow are only intended for the purposes of illustration, and are notto be construed as limiting in any manner to make compounds of thedisclosure by other methods.

EXAMPLES

Clinical data and banked plasma specimens were combined from an ongoingNHLBI-sponsored multinational multicenter trial of VTE treatment inpatients <21 years old (the Kids-DOTT trial) with those from asingle-institutional prospective cohort study of VTE in patients <21years old at Johns Hopkins All Children's Hospital (St. Petersburg,Fla., USA). The CloFAL global assay was performed as previouslydescribed, on banked plasma samples from 4-6 weeks and 3 monthspost-diagnosis of acute VTE, with heparin pre-treatment of samples priorto assay.

Study Design

This study combined clinical data and banked plasma biospecimenscollected at 4-6 weeks and 3 months post-diagnosis of provoked VTE inthe pediatric VTE cohort of the Johns Hopkins All Children's (St.Petersburg, Fla.) institution-wide prospective inception multi-cohortand biobanking study (iPICS) with that from the NHLBI-sponsoredKids-DOTT trial. In many cases, these two time points simultaneouslyrepresented 6 weeks+/−2 weeks prior to planned cessation ofanticoagulation and time of planned cessation (or within 2 weekspost-cessation) of anticoagulation, respectively. The study was approvedby the local Institutional Review Board at each participating site, withsigned informed consent/assent required for patient participation.

The Kids-DOTT trial design has also been previously reported in detail(Goldenberg N A, Abshire T, Blatchford P J, Fenton L Z, Halperin J L,Hiatt W R, et al. Multicenter randomized controlled trial on Duration ofTherapy for Thrombosis in Children and Young Adults (the Kids-DOTTtrial): pilot/feasibility phase findings. J Thromb Haemost. 2015;13(9):1597-605; Bernard T J, Armstrong-Well J, Goldenberg N A. Theinstitution-based prospective inception cohort study: design,implementation, and quality assurance in pediatric thrombosis and strokeresearch. Semin Thromb Hemost. 2013; 39(1):10-14). The present studyincluded patients enrolled in the iPICS VTE cohort from 2013 through2016 and in Kids-DOTT from 2007 through 2016.

Blood Sample Collection, Processing, and Preparation for LaboratoryAnalyses

Whole blood was collected at follow-up visits 4-6 weeks and 3 monthspost-diagnosis of VTE via atraumatic peripheral venipuncture usingminimal applied stasis into BD Vacutainer 3.2% buffered sodium citratesiliconized blood collection tubes. Sample processing occurred within 1hour from collection. Samples were centrifuged at 2500 g for 15 minutesat 4° C., afterwards the supernatant was isolated and subjected torepeat centrifugation for 15 minutes at the aforementioned settings. Theresulting platelet-poor plasma supernatant was pooled, aliquoted intopropylene tubes, and frozen at −80.0 in SAM units (Hamilton®, Reno,Nev., USA) of the College of American Pathologists-accredited JHACHPediatric Biorepository for long-term storage until time of assay. Allplasma aliquots undergoing CloFAL assay analysis were heparinase-treatedonce thawed in preparation for the assay.

Kids-DOTT Trial Assessments

Study participation was expected to last for 2 years. Many of theassessments in this study were completed as part of the standard of carevisits. The study comprised the following: Review of scans of the bloodclot performed at diagnosis and at approximately 6 weeks afterdiagnosis; Randomization to either 6 weeks or 3 months duration of bloodthinner treatment; Blood sample collection for research at 6 weeks and 3months post-diagnosis; Brief follow-up phone calls at 2, 4 and 5 monthsfollowing diagnosis; In-person follow-up at 6 weeks, and 3, 6, 12, and24 months, after diagnosis; Treatment diary to track when doses of theparticipants blood clot medication are missed; Tracking of medicalevents that happen to the participant;

Study Participation Requirements: Recently diagnosed, for the firsttime, with an acute venous thrombosis; Age less than 21 years old attime of diagnosis of blood clot; Planned (or already on) treatment forthe acute blood clot; No history of cancer; No prior history of treatedblood clots.

Those few patients (n=3) who received agents other than low molecularweight heparin at these time points were excluded. Assay measurementsincluded maximal amplitude of the CloFAL waveform (MA), time to maximalamplitude (T1), and area under the curve at 60 minutes, indexed to thatof the pooled normal plasma standard (AUC₆₀). For each patient, thechange in each CloFAL parameter between the 3 months and the 4-6 weekpost-VTE time points was then calculated.

Statistical Analyses

Descriptive statistics were used to summarize data on patientcharacteristics and outcomes. For each patient, the percentage change ineach CloFAL parameter (MA, T1, AUC₀₋₆₀ min and FI₂) was calculatedbetween the 3 month and the 4-6 week sample post-diagnosis of index VTE.We planned to employ receiver operating characteristics (ROC) curves andYouden's J index to establish putative thresholds to dichotomize percentchange for each CloFAL parameter. If this approach was non-informative,we planned to use a threshold of 50% change (specifically, ≥50% increasefor MA and AUC0-60 min, and a ≥50% decrease for T1 and FI₂),conservatively selected to assure that this threshold was substantivelygreater than the inter-assay coefficient of variation of the assay foreach parameter.

Associations with VTE recurrence for the aforementioned dichotomizedvariables for marked subacute changes in CloFAL parameters and forelevated D-dimer levels at 3 months were determined by univariablelogistic regression with Firth's penalized likelihood approach, toobtain odds ratios (OR) and 95% confidence intervals (CIs). For thisaim, patients in whom VTE recurrence occurred before 3 monthspost-diagnosis of the index VTE (n=1) were excluded from the analysis.Sensitivity was also calculated from the proportion of patients withrecurrent VTE in which the prognostic laboratory variable of interestwas positive. Specificity was defined as the proportion of patientswithout recurrent VTE in which the laboratory determinant was negative.

Positive likelihood ratios were calculated assensitivity÷(1−specificity). Change in pre-test to post-test probabilityof recurrent VTE was calculated using the positive likelihood ratio, aspreviously described (Goldenberg N A, Knapp-Clevenger R, Manco-Johnson MJ, et al. Mountain States Regional Thrombophilia Group. Elevated plasmafactor VIII and D-dimer levels as predictors of poor outcomes ofthrombosis in children. N Engl J Med. 2004; 351(11):1081-8). Allstatistical analyses were performed using SAS v 9.4 and Stata v 15, withstatistical significance defined as a two-sided alpha <0.05.

Descriptive statistics were used to summarize data on patientcharacteristics, VTE presentation, treatment, and outcomes. To exploreprognostic cutoffs for percent change in each parameter receiveroperating characteristics curves were generated, and inter-assaycoefficients of variation of the assay were also considered. Thresholdsof 50% increase in MA or AUC₆₀, or 50% decrease in T1, were ultimatelyselected as candidate phenotypes of relative rebound hypercoagulability.An association between marked rise in plasma coagulability during thesubacute period post-VTE and VTE recurrence was determined viaunivariate logistic regression, using odds ratios (OR) and 95%confidence intervals (CI), with two-sided alpha=0.05. The blind wasmaintained in the Kids-DOTT trial throughout data transfer and analysis.

Results: The final study population consisted of 88 patients, with amedian age of 12.6 years (range, 0.01-20.8 years). VTE was classified asprovoked in 98% of cases. Patient and VTE characteristics at baselineand outcomes during follow-up are shown in Table 1. Median follow uptime was 52 weeks (range 9-123 weeks). A marked rise in plasmacoagulability during the subacute period post-diagnosis of VTE was foundin 13% of the study population. Recurrent VTE developed in 10% ofpatients. Logistic regression analysis revealed that change in CloFALAUC₆₀ and T1 were not prognostic of VTE recurrence. However, a markedrise in plasma coagulability during the subacute period post-diagnosisof VTE, as measured by change in CloFAL MA, was associated with astatistically significant, 4-fold increase in the risk of recurrent VTE(OR=4.47, 95% CI=1.05-19.0; P=0.04).

TABLE 1 Patient and VTE characteristics at baseline and outcomes duringfollow-up. Patients (n = 88) Median age (range)   12.6 (0.01-20.8)Gender female no. (%) 49 (55.6) Location Index VTE no. (%) UpperExtremity 28 (31.8) Lower Extermity 37 (42.0) CSVT 12 (13.6) Other* 11(12.5) Type of VTE no. (%) Provoked 87 (98.8) Unprovoked 1 (1.2)CVC-associated VTE no. (%) 48 (54.5) Recurrent VTE no. (%)  9 (10.2)Marked subacute rise in 12 (13.6) coagulability** no. (%) Median Followup weeks  52 (9-123) (range) Abbreviations: VTE: venous thromboembolism;CSVT: cerebral sinovenous thrombosis; CVC: central venous catheter.*Other: included internal jugular vein only, superior vena cava only,and splanchnic vein only. **As defined by the number of patientsexhibiting a 50% or greater increase in MA in the CloFAL assay at 3months relative to 4-6 weeks post-diagnosis of VTE.

FIG. 1 provides ROC curves for subacute change (measurements at 3 monthsvs. 4-6 weeks post-diagnosis of index VTE) in each CloFAL assayparameter of interest. C-indices were as follows: AUC, 0.65; MA, 0.71;T1, 0.66; FI₂, 0.44. Using Youden's J index, the following prognosticthresholds for subacute change in CloFAL parameters were initiallydetermined: increase in AUC, 10.3%; increase in MA, 10.0%; decrease inT1, 1.4%; decrease in FI₂ 1.1%. Because each of these derived thresholdsfell within the corresponding inter-assay coefficient of variation forCloFAL assay parameters, the secondary approach of prognostic thresholdevaluation was employed, in which a marked subacute rise incoagulability was set at a 50% change in MA, AUC, or T1, and a markedsubacute decline in fibrinolytic capacity was set at a 50% change inFI₂.

Table 2 shows the distribution of CloFAL parameters and theirassociation with recurrent VTE. D-dimer was elevated at 3 months in 18patients (20.9%). By comparison, a marked subacute rise in coagulability(see Methods) was observed in 15 patients (17.4%) by CloFAL MA, in 14patients (16.2%) by AUC₀₋₆₀ mm, and in only one patient (1.1%) by T1.Two patients (2.3%) showed a marked subacute decline in fibrinolyticcapacity, as measured by CloFAL FI₂. FIG. 2 shows representative CloFALcurves of patients with and without a marked subacute rise incoagulability by CloFAL MA and AUC. There was no significant associationbetween elevated D-dimer levels at 3 months and subacute rise incoagulability as measured by ≥50% increase in MA or AUC (Fisher's exactP=0.29, and P>0.99, respectively).

Univariable logistic regression analyses (Table 2) revealed that amarked subacute rise in coagulability as defined by CloFAL MA wasassociated with a statistically-significant, six-fold increase (OR=5.87,95% CI=1.34-25.8; P=0.019) in the odds of recurrent VTE. Neither thesubacute change in CloFAL AUC, T1, or FI₂, nor an elevated D-dimer levelat 3 months, was significantly associated with the risk of recurrentVTE. Similarly, neither a ≥50% increase in D-dimer levels at 3 monthsrelative to 4-6 weeks post-VTE diagnosis, nor a change in D-dimer fromnegative at 4-6 weeks to positive at 3 months, were associated with anincreased risk for recurrent VTE (Table 2).

A marked subacute rise in coagulability by CloFAL MA was observed in 22%of patients who were receiving anticoagulation, and in 13% of patientswho were not receiving anticoagulation, at 3 months post-diagnosis ofthe index VTE. We determined a positive likelihood ratio of 4.73 (95% CI1.33-16.8) for the association of a subacute rise in coagulability byCloFAL MA with the subsequent development of recurrent VTE. Given thislikelihood ratio, and a pre-test probability of recurrent VTE of 10.3%(i.e., prevalence in the study population), the post-test probability ofrecurrent VTE in the setting of provoked VTE with a subacute rise incoagulability by CloFAL MA was 35.2%.

TABLE 2 Marked rise in coagulability in the subacute periodpost-diagnosis of VTE, D-dimer and risk of recurrent VTE Frequency inMeasure of Putative Study Association with Prognostic PopulationRecurrent VTE Factor* n (%) OR 95% CI P-value CloFAL MA 15 (17.2) 5.871.34-25.8 0.019 CloFAL_(AUC0-60 min) 14 (16.1) 3.74 0.82-17.0 0.089CloFAL T1  1 (1.1) 3.01  0.03-297.3 0.638 CloFAL FI₂  2 (2.3) 1.8 0.04-79.4 0.761 Elevated D-dimer 10 (20.7) 1.46 0.30-7.15 0.643 at 3mos. Abbreviations: VTE, venous thromboembolism; CI, confidenceinterval; MA, maximum amplitude; AUC, area under the curve; T1, time tomaximum amplitude; FI2, modified fibrinolytic index. *CloFAL parameterswere evaluated for subacute change (3 month vs. 4-6 week measurements).† Elevated D-dimer was defined as >0.42 μgFEU/mL

Conclusions: A marked rise in plasma coagulability during the subacuteperiod post-diagnosis of VTE, as measured by change in MA in the CloFALassay, developed in 13% of a multicenter study population of patients<21 years of age with predominantly-provoked VTE, and was associatedwith a 4-fold increased risk of recurrent VTE. Future work will seek toinvestigate mechanisms underlying the rise in plasma coagulabilityduring the subacute phase post-diagnosis of VTE in young patients.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method for diagnosing risk of rise in plasma coagulability in asubject who previously experienced a thrombotic event (TE) and hasundergone anticoagulation therapy, comprising the steps of: a) analyzinga first biological sample taken from a subject at time period of about 4to about 6 weeks post-diagnosis of TE, using the CloFAL assay method andgenerating a CloFAL MA value; b) analyzing a second biological sampletaken from a subject at time period of about 3 months post-diagnosis ofTE using the CloFAL assay method and generating a CloFAL MA value; c)comparing the CloFAL maximum amplitude value of a) to b); and d)identifying the subject as being at risk for a rise in plasmacoagulability when the CloFAL MA value of b) is at least 50% greaterthan the CloFAL MA value of a).
 2. The method of claim 1, wherein thesecond biological sample is taken from a subject at time period of about6 months post-diagnosis of TE.
 3. The method of claim 1, wherein thesecond biological sample is taken from a subject at time period ofgreater than 6 months post-diagnosis of TE.
 4. The method of claim 1,wherein the biological sample is a blood sample. 5.-8. (canceled)
 9. Amethod for identification or diagnosis of who is at risk for a markedrise in plasma coagulability and recurrent thromboembolism in a subjectwho previously experienced a thrombotic event (TE) and underwentcoagulation therapy comprising the steps of: a) analyzing a firstbiological sample taken from a subject at time period of about 6weeks+/−2 weeks prior to planned cessation of coagulation therapy usingthe CloFAL method and generating a CloFAL maximum amplitude value; b)analyzing a second biological sample taken from a subject at time ofplanned cessation (or within about 2 weeks post-cessation) ofanticoagulation therapy using the CloFAL method and generating a CloFALmaximum amplitude value; c) comparing the CloFAL maximum amplitude valueof a) to b); and d) identifying the subject as having a marked rise inplasma coagulability and therefore as being at heightened risk forrecurrent thromboembolism when the CloFAL maximum amplitude value of b)is at least 50% greater than the CloFAL maximum amplitude value of a).10. The method of claim 9, wherein the second biological sample is takenfrom a subject at time period of about is at least about 1 month toabout 24 months post-cessation of coagulation therapy.
 11. The method ofclaim 10, wherein the second biological sample is taken from a subjectat time period at least about 3 months post-cessation of coagulationtherapy.
 12. The method of claim 9, wherein the biological sample is ablood sample. 13.-15. (canceled)